Metabolic engineering of microbial pathways provides a cost-effective and environmentally benign route for producing numerous valuable compounds, including commodity and specialty chemicals (e.g. biodegradable plastics), biofuels (e.g. ethanol and butanol) and therapeutic molecules (e.g. anticancer drugs and antimicrobial compounds). However, efforts to engineer new functional biosynthetic pathways in well-characterized micro-organisms such as Escherichia coli are still often hampered by issues such as imbalanced pathway flux, formation of side products and accumulation of toxic intermediates that can inhibit host cell growth. One strategy for increasing metabolite production in metabolically engineered microorganisms is the use of directed enzyme organization [for a review see Conrado et al., “Engineering the Spatial Organization of Metabolic Enzymes Mimicking Nature's Synergy,” Curr. Opin. Biotechnol. 19:492-499 (2008)]. This concept is inspired by natural metabolic systems, for which optimal metabolic pathway performance often arises from the organization of enzymes into specific complexes and, in some cases, enzyme-to-enzyme channeling (a.k.a. metabolic channeling) (Conrado et al., “Engineering the Spatial Organization of Metabolic Enzymes: Mimicking Nature's Synergy,” Curr. Opin. Biotechnol. 19:492-499 (2008); Srere P. A., “Complexes of Sequential Metabolic Enzymes,” Annu. Rev. Biochem. 56:89-124 (1987); Miles et al., “The Molecular Basis of Substrate Channeling,” J. Biol. Chem. 274:12193-12196 (1999)).
The most striking naturally occurring examples are enzymes that have evolved three-dimensional structures capable of physically channeling substrates such as tryptophan synthase and carbamoyl phosphate synthase. The crystal structures of these enzymes reveal tunnels that connect catalytic sites and protect reactive intermediates from the bulk solution (Hyde et al., “Three-Dimensional Structure of the Tryptophan Synthase α2β2 Multienzyme Complex From Salmonella typhimurium,” J. Biol. Chem. 263:17857-17871 (1988); Thoden et al., “Structure of Carbamoyl Phosphate Synthetase: A Journey of 96 A From Substrate to Product,” Biochemistry 36:6305-6316 (1997)). Other notable examples include electrostatic channeling of negatively charged substrates along a positively charged protein surface that leads from one active site to the next (Stroud R. M., “An Electrostatic Highway,” Nat. Struct. Biol. 1:131-134 (1994)), direct channeling of substrates via thioester linkages between polyketide synthase enzyme modules (Tsuji et al., “Selective Protein—Protein Interactions Direct Channeling of Intermediates Between Polyketide Synthase Modules,” Biochemistry 40:2326-2331 (2001)), compartmentalization of specific enzymes into small volumes within the cell in the form of subcellular organelles (Bobik T. A., “Polyhedral Organelles Compartmenting Bacterial Metabolic Processes,” Appl. Microbiol. Biotechnol. 70:517-525 (2006); Straight et al., “A Singular Enzymatic Megacomplex From Bacillus subtilis,” Proc. Nat'l. Acad. Sci. U.S.A. 104:305-310 (2007)), and dynamic assembly of enzyme complexes, perhaps as a feedback mechanism, to achieve a precise concentration of metabolic product (Narayanaswamy et al., “Widespread Reorganization of Metabolic Enzymes Into Reversible Assemblies Upon Nutrient Starvation,” Proc. Nat'l. Acad. Sci. U.S.A. 106:10147-10152 (2009); An et al., “Reversible Compartmentalization of de Novo Purine Biosynthetic Complexes in Living Cells,” Science 320:103-106 (2008)).
Inspired by these natural systems, several groups have developed methods for artificially assembling enzyme complexes to enhance the performance of biological pathways. For example, direct enzyme fusions have been used to coordinate the expression and localization of two resveratrol biosynthetic enzymes in a manner that increased product titers in yeast and mammalian cells (Zhang et al., “Using Unnatural Protein Fusions to Engineer Resveratrol Biosynthesis in Yeast and Mammalian Cells,” J. Am. Chem. Soc. 128:13030-13031 (2006)). However, fusing more than two enzymes may prove problematic due to misfolding and/or proteolysis of the fusion protein. In a notable departure from fusion proteins, Fierobe and co-workers constructed artificial cellulosomes where selected enzymes were incorporated in specific locations on a protein scaffold (Fierobe et al., “Design and Production of Active Cellulosome Chimeras. Selective Incorporation of Dockerin-Containing Enzymes Into Defined Functional Complexes,” J. Biol. Chem. 276:21257-21261 (2001)). Compared to their free enzyme counterparts, the resulting enzyme complexes exhibited enhanced synergistic action on crystalline cellulose. More recently, Dueber et al., “Synthetic Protein Scaffolds Provide Modular Control Over Metabolic Flux,” Nat. Biotechnol. 27:753-759 (2009) expressed scaffolds built from the interaction domains of metazoan signaling proteins to assemble metabolic enzymes that were tagged with their cognate peptide ligands. Significant increases in the production of mevalonate and separately glucaric acid were observed in the presence of several of these scaffolds. Along similar lines, Delebecque et al., “Organization of Intracellular Reactions With Rationally Designed RNA Assemblies,” Science 333:470-474 (2011) created RNA aptamer-based scaffolds to control the spatial organization of two metabolic enzymes involved in biological hydrogen production. Similar to protein scaffolds, RNA-based scaffolds increased the hydrogen output as a function of scaffold architecture.